Nucleotide co-delivery modulates intracellular fate of LNP-delivered mRNA to enhance translation

Messenger RNA (mRNA) therapeutics offer a versatile platform for protein production, but preserving mRNA during delivery remains a major challenge. Here, we report that co-delivery of nucleotides with lipid nanoparticle (LNP)-formulated mRNA significantly enhances translation efficiency across multiple cell types. Systematic screening identifies adenosine triphosphate (ATP) and guanosine triphosphate (GTP) as effective enhancers, increasing protein expression by up to 13-fold, accompanied by ~6–8-fold increases in intracellular intact mRNA levels. The non-hydrolyzable nucleotide analogues adenylyl imidodiphosphate (AMP-PNP), guanylyl imidodiphosphate (GMP-PNP) retain comparable expression-enhancing activity, suggesting that the effect does not require phosphate-bond hydrolysis or canonical ATP/GTP-dependent energy supply. Mechanistic analyses show largely unchanged cellular uptake and endosomal escape under the tested conditions. Nucleotides delay the degradation of LNP-delivered mRNA in culture medium, protect purified mRNA against RNase A-mediated degradation, and reduce RNA-substrate cleavage in purified-enzyme and cell-lysate assays. Nucleotide co-encapsulation also improves reporter expression in mice following intramuscular administration. Together, these findings support mRNA preservation as a contributor to enhanced expression and suggest a transient protective environment during early delivery. This study provides mechanistic insights into nucleotide-assisted mRNA delivery and a basis for further development of mRNA vaccines and protein replacement therapies.